Ion Exchange Technique for the Determination of Chlorinated Phenols and Phenoxy Acids in Organic Tissue, Soil, and Water Lars Renberg National Swedish Environment Protection Board, Special Analytical Laboratory, Wallenberg Laboratory, Lilla Frescati, 104 05 Stockholm 50, Sweden
The fungicidal properties of pentachlorophenol, 2,3,4,6tetrachlorophenol and 2,4,6-trichlorophenol have been utilized for wood protection, by dipping or pressure impregnation, and for slime control in the wood industry and paper mills. Hexachlorophene (2,2’-methylene-bis(3,4,6trichlorophenol)) and 2-hydroxy-2’,4,4’-trichlorodiphenyl ether are commonly used as bactericides. As herbicides, the 2,4-dichlorophenoxy acetic acid and 2,4,5-trichlorophenoxy acetic acid are often used, mainly against weeds. All these compounds are relatively stable because of the chlorinated aromatic ring structure, but the polar hydroxy and carboxy groups tend to facilitate the biological degradation. The ability of these compounds to form anions is utilized in the analytical method described below. As the use of these substances has been reported to cause negative effects on man and his environment ( I , 2), the need for analyzing the compounds at a low level has resulted in the publication of many different methods. Because of the high sensitivity requirement, the analyses are usually carried out by gas chromatography using an electron capture detector (ECD) but other methods, such as spectrophotometry (3. 4 ) have also been used. Usually the polar hydroxy-and carboxy-groups are converted into the ether or ester derivatives before the gas chromatographic analysis. The most common derivatives are the methyl ethers ( I , 5 ) , methyl esters (6, 7), and the trimethylsilyl ethers (5, 7). Diazomethane is used as the methylation agent in the method described here. In the case of the phenoxy acetic acids, a new derivation procedure is developed. Besides the methyl esters, the 2-chloroethyl esters are also prepared. Several papers have dealt with a clean-up using partition of the substances between a lipophilic and a hydrophilic liquid phase under alkaline and acidic conditions. High fat contents (in order to lower the detection limit) and especially fat of marine origin have been shown to seriously affect the possibility to separate the two liquid phases. Another problem arises when analyzing soil samples. When the alkaline extract is acidified, gel formation often occurs at pH values lower than 6. This has been shown to interfere with the extraction of pentachlorophenol ( 5 ) .As this type of substances usually has pK, values below 6, this can make the extraction difficult or impossible. These problems can be avoided by binding the acidic substances under alkaline condition to an anion ion exchanger. The liquid phases can then be discharged. The substances to be studied are later removed from the ion exchanger under acidic conditions. (1) B. Holrnberg, S. Jensen, A. Larsson, K. Lewander, and M . Olsson. Comp. Biochem. Physiol., 43 171 (1972). (2) J. D. Lockhart, Pediatrics, 50229 (1972). (3) V. D.Johnston and P. J . Porcaro, Anal. Chem., 36 124 (1964). (4) P. J. Porcaro and P. Shubiak. Anal. Chem., 44 1865 (1972). (5) A. Stark, J. Agr. FoodChem., 17871 (1969). (6) L . Rudling, WaterRes., 4 , 5 3 3 (1970). ( 7 ) A . G. Ulsamer, J. Ass. Offic. Anal. Chem., 55 1294 (1972).
Ion exchange can be carried out in a column or by a batch procedure. When equilibrium conditions allow a batch procedure to be used, it usually offers a fast and simple technique. This has been applied in the analysis of organic tissue and soil. When analyzing water samples on the other hand, the column procedure is used, as it allows large amounts (no principal limit) of water to pass through, to determine very low levels.
EXPERIMENTAL Reagents and Equipment. Hexane, acetone, diethyl ether (anhydrous), methanol, potassium chloride (0.2M), sodium hydroxide (0.2M and O.lM), hydrochloric acid (1.OM and 0.2M), sodium sulfate (anhydrous), 2-chloro-ethanol, diazomethane in diethyl ether solution ( 8 ) , Sephadex QAE, A-25 anion exchanger. All reagents should be tested in a blank procedure. An acidic buffer was prepared by mixing equal volumes of the hydrochloric acid ( 0 . 2 M and potassium chloride solutions. The ion exchanger is swollen in distilled water a t least two hours before use. A 10-cm X 1-cm (i.d.) column was used when analyzing water samples. For the batch procedures, 15-ml test tubes with screw caps and Teflon (Du Pont) packings were used. A Varian 1400 gas chromatograph, equipped with ECD (tritium) was used. The 160 cm x 0.18-cm (i.d.) glass columns were SF 96 (lye), or a mechanically prefilled with either OV-17 (l’70), pared mixture of 67 parts QF 1 (8%) and 33 parts of SF 96 (4%) on acid-washed, silanized Chromosorb W 100/120 mesh. The column temperature and the corresponding relative retention times are shown in Table I. Injector and detector temperatures were held about 10°C above the column temperature. Procedures. Analysis of Organic Tissues. Step 1. Homogenize the sample (5 grams) in a mixture of hexane and acetone (5 + 10 ml) by means of an insertable homogenizer in a dropping funnel with a glass filter disk. Drop the liquid into a separatory funnel containing 1.OM hydrochloric acid (5 ml); use nitrogen pressure if necessary. Homogenize once more with a mixture of hexane and diethyl ether (10 5 ml) and collect the mixture in the separatory funnel. Shake the funnel and transfer the upper phase into a centrifuge tube, then re-extract the water phase twice with a mixture of diethyl ether and hexane (2 2 ml) and transfer the extracts to the centrifuge tube. Add sodium sulfate (100-300mg) to bind any water present. After centrifugation, transfer the extract into a weighed flask, rinse the sodium sulfate with diethyl ether ( 2 ml) and evaporate the solvents gently on a water bath in a nitrogen stream. Weigh the flask again and calculate the fat content; then dissolve the fat in benzene (use about 1 m1/25 mg fat). Step 2. Transfer the suspension of the ion exchanger into a 15-ml test tube and, after centrifugation, discard the water. The bed volume should be about 3 ml. Add to the test tube 3 ml of the benzene solution and 3 ml of sodium hydroxide solution (0.1M). Shake the test tube carefully for 5 min, and remove the liquid phases after centrifugation (the benzene phase can be used for analysis of nonacidic pesticides). Add to the ion exchanger 3 ml of distilled water, shake the test tube for about 30 seconds, and, after centrifugation, discard the water. Step 3. Add 3 ml of benzene (containing a suitable internal standard) and 3 ml of the acidic buffer. Shake carefully for about 5 min and transfer the benzene phase into a graduated test tube. Convert the substances into suitable derivatives as described under the preparation of derivatives below. Analysis of Water Samples. Transfer a suspension of the ion
+
+
~~
~
~~~
~
Table I. Levels of Substances in Fortified Samples and Corresponding Recoveries Soil, 1 gram
Water, loo0 ml
Fish tissue, 5 grams
Level, ppb
Rec., yo
Level, ppm
0.50
>97 >97 >97
0.50 0.50
0.30
74 90 92
1.0
79
5
94
1.0
83
5
92
3.2 1.6
70 82
16
>97 >97
Level, ppm
Fungicides 2,4,6-Trichlorophenol 2,3,4,6-Tetrachlorophenol Pentachlorophenol Bactericides 2-Hydroxy-2',4,4'trichlorodiphenyl ether Hexachlorophen Herbicides 2,4-D-acid 2,4,5-T-acid
0.10 0.10
Rec., %
0.50 1.5
8.0
Humus rec., %
1.5
>97 >97 >97
...
...
16
8.0
70 86
Clay rec., %
>97 >97 >97
74 84
The ion exchange conditions chosen here allow one to analyze many kinds of organic substances, capable of forming water soluble anions. With other buffers than those described here, the method can be made more specific for a particular substance. The ion exchanger chosen for these experiments is a strongly basic anion exchanger. The functional group is diethyl-(hydroxy-propyl) amino ethyl, with chloride as the counter ion. The capacity of the described batch proce-
dure was a t least 0.1 mg/ml of swollen ion exchanger (using an ion strength of O.lM), with respect to the studied substances. The described extraction method has been developed particularly for acidic pesticides. Other well tested extraction methods can naturally be used. However, there are two steps in an extraction procedure for this type of substances, which have to be carefully considered. First, extraction solvent mixtures usually consist of a nonpolar solvent such as hexane or heptane and a dehydrating solvent, for example acetone or isopropyl alcohol, which serves to open the cells for extraction. This latter type of solvent partly dissolves the water from the tissue sample. This results in a two-phase system, where the relatively polar pesticides are partitioned between the phases. Too much acetone in the water phase and a too high p H value will result in an unfavorable partition (especially for the phenoxy acids) for the lipophilic phase. This problem can be overcome either by removing the water by means of a drying agent, or by acidifying the water, which will result in a change of equilibrium in favor of the organic phase. Second, the fat extract is evaporated to dryness to determine the fat content of the sample. Normally, pesticide contents are related to the fresh tissue but from an ecological point of view, calculations on the fat weight basis are valuable. The evaporation step could result in a loss of substances. However, evaporation with the use of a nitrogen stream and a water bath have been shown to be satisfactory, even for the volatile phenols (as long as fat is present in the extract). As described. soils were extracted twice with sodium hydroxide solution. In contrast to Stark ( 5 ) , one extraction of humus soil was found not to be efficient. Recovery experiments with fish tissues were carried out with the substances added before extraction. Also butter and eel fat, representing different degree of unsaturation were spiked and the recovery studied. No interference from free fatty acids occurred. When testing the recoveries from soil and water, the substances were added in ethanol solutions. Two different kinds of soil (clay and humus soil) were used, representing different p H values. The recoveries are shown in Table I. For the gas chromatographic determination, three different types of stationary phases were used. The OV-17 is a generally used low bleeding stationary phase. This makes it suitable for combined gas chromatography-mass spectrometry (especially mass fragmentography). The mixture of SF 96 and QF 1 is specially designed for analysis of DDT and PCB (9). When hexachlorophen and heav-
(8) Th. J . de Boer and H. J . Backer, R e d Trav. Chim. Pays-Bas. 73,
(9) S
exchanger into the column (see equipment above). Let the ion exchanger settle and connect the upper end of the column to a separatory funnel containing the water sample. The bed volume should be about 3-4 ml. If an increase of the outflow is desired, connect the lower end of the column to a water suction pump (without a pump the outflow usually varies between 0.6-0.8 ml/ min). Two different methods can now be used for eluting the substances from the ion exchanger. Alternative 1. Add 3-4 ml of distilled water into the column, shake it carefully and decant the suspension into a 15-ml test tube. Wash the column with another portion of water to ensure that all ion exchanger is transferred to the test tube. After centrifugation, remove the water and continue according to step 3 above. Alternative 2. Elute the column with 10 ml of acidified methanol (1 gram sulfuric acid/50 ml methanol). Add to one part of the eluate (in a test tube) an equal volume of benzene and 4 parts of the hydrochloric acid solution (1.OM). Shake the test tube and, after centrifugation, transfer the benzene phase into a graduated test tube and convert the substances into suitable derivatives as described under the preparation of derivatives below. Anal>sis of Soil Samples Shake the sample with 0.2M sodium hydroxide (4 ml/gram soil) in a test tube, for 30 min. After centrifugation remove the liquid and re-extract with a new portion of sodium hydroxide solution. Estimate the volume of the combined alkaline extracts. Shake 2 ml of the extract and 8 ml of water for 10 min with the ion exchanger (3-ml bed volume). After centrifugation, discard the liquid and rinse the ion exchanger with 5 ml of distilled water. Discard the water and continue as described in step 3 above. Preparation of Derimtives Methyl Ethers of the Phenols and Methyl Esters of the Phenoxy Acetic Acids. Prepare diazomethane in ether solution from e g. N-methyl-N-nitroso-p-toluenesulfonamide (8). Add the diazomethane solution to the benzene extract until the extract becomes pale yellow. After about 1 hour, evaporate to the original volume. Inject the extract into the gas chromatograph and compare the result with a standard treated the same way. 2-Chloroethyl Esters of the Phenoxy Acetic Acids. Shake a 15-ml test tube containing 2 ml of the benzene extract, 1 ml of 2chloro ethanol and 100 ~1 sulfuric acid for 1 min, and let stand in a water bath a t 50 "C for 30 min. Then add 10 ml of distilled water, shake, and centrifuge. Inject the benzene phase into the gas chromatograph and compare the result with a standard treated in the same way.
RESULTS AND DISCUSSIONS
229 (1954).
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Jensen A G Johnels Rep 1 7 1 (1972)
M
Olsson, and G Otterlind Arnbio Spec
Table 11. Retention Times Relatively -/-BHC, p,p-DDE, and p,p-DDT
7-BHC
Methyl ethers of 2,4,6-Trichlorophenol 2,3,4,6-Tetrachlorophenol Pentachlorophenol r-BHC
pp-DDE Methyl esters of 2,4-Dichlorophenoxy acetic acid 2,4,5-Trichlorophenoxy acetic acid 2-Chloroethyl esters of 2,4-Dichlorophenoxy acetic acid 2,4,5-Trichlorophenoxy acetic acid Methyl ether of 2-hydroxi-2 ',4,4 '-Trichlorodiphenyl ether
+
OV-17 7 rnin at 160 o c
QF1 SF 96 9 min at 150 OC
0.096 0.64
0.14 0.32 0.70
1 min at 200 o c
3 min at 180 o c
2.93
3.02
1.24 1.83
0.94 1.29
1.70 2.95
2.65 4 .OO
2.93
3.27
Dimethyl ether of Hexachlorophen
In fresh tissue
1. Pike 0.25
muscle liver gills 2. Pike muscle liver gills 3. Eel muscle 4. Ide muscle 5. Burbot muscle 6. Roach muscle
In extractable fat
12 23 19
1200 200 650
9.5 25 26 0.35 6.2 7.8 6.0
1100
160 710 1.9 350 1100
580
Water sample, taken two days after the discharge, contained 0.35 ng/ml.
SF 96 pp-DDT
Table 111. Levels of Pentachlorophenol (ppm) in Water and Fish Found Dead in a Contaminated Rivera
2 min at 200 o c
3.42
ier derivatives of the other chlorinated compounds are to be analyzed, the 1% SF 96 is preferable, because of short retention times a t relatively low temperatures. The use of internal standard in the gas chromatographic determination is recommended. Y-BHC (lindane), DDE, or DDT are suitable; the choice depends on the kind of substances to be studied. The relative retention times of the derivatives corresponding to these internal standards are shown in Table 11. The detection limits for the differ-
ent substances in 10 grams of an organic tissue or soil are 0.1-1 ppb and for one liter of a water sample 0.001--0.1 PPb. The method was tested on fish found dead in a river in the south of Sweden where a discharge of pentachlorophenol was suspected. Also a water sample, taken two days later was analyzed. The levels of pentachlorophenol were in accordance with those found to be lethal for the eel ( 1 ) . The results are shown in Table 111.
ACKNOWLEDGMENT The author is greatly indebted to Soren Jensen, head of the laboratory, for his valuable advice and constructive criticism of the manuscript. Received for review August 6, 1973. Accepted October 30, 1973.
I CORRESPONDENCE Hollow Cathode Ion Source for Solids Mass Spectrometry Sir: Hollow cathode discharge tubes are well known as sharp line spectral sources which have long been useful in physics ( I , 2) and more recently as source lamps for atomic absorption spectrometry. Demountable hollow cathode tubes may be utilized as atomic emission sources for trace element analysis (3-6) by placing the solid sample in the cathode or by evaporating a solution sample in the cathode cavity. The surface sputtering action of the hollow cathode discharge yields excellent elemental sensitivity (7) by moni(1) H. Schuler and H.Goiinow, 2. Physik, 93,611 (1935). (2) A . G.Shenstone. Trans. RoyalSoc. (London), A235, 195 (1936). (3) J. R . McNaily. G. R . Harrison, and E. Rowe, J. Opt. SOC.Amer. 37, 93 (1947) (4) G.Milazzo and N. Sopranzi. Appl. Spectrosc., 21,256 (1967). (5) W. W. Harrison and N. J. Prakash, Anal. Chim. Acta. 49, 151 (1970). (6) N. J. Prakash and W . W. Harrison, Anal. Chim. Acta, 53, 421 (1971 ) . (7) W. W. Harrison and E. H. Daughtrey, Anal. Chim. Acta, 65, 35 (1973).
toring the excited atomic species. A large neutral population is, of course, also formed.' It is further known, from ion microprobe mass spectrometry, that ion bombardment of a surface can produce secondary ions of the target species. Coburn and Kay (8, 9 ) have shown that both dc and rf discharges may be used in a planar diode sputtering system to determine major constituents in a surface film. Our initial intent in interfacing a hollow cathode tube to a mass spectrometer was to gain a better understanding of the reactions and plasma conditions which exist in our hollow cathode emission source. Subsequent experiments have shown that the hollow cathode discharge may offer advantages as a mass spectrometry surface ionization source for solids, such as metals and alloys. It can also be used to provide survey analysis of the trace elements in a (8) J. W. Coburn, Rev. Sci. lnsfrum., 41,1219 (1970) (9)J. W. Coburn and Eric Kay, Appl. Phys. Lett., 19,350 (1971).
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